The metal binding sites of a large number of metalloproteins contain imidazole rings from histidyl residues of the protein. One metalloprotein that contains a large number of histidyl residues in its active site and which has interesting physical and enzymatic properties is copper-zinc superoxide dismutase. This enzyme, of molecular weight 32,000, has two equivalent subunits which each bind one copper and one zinc ion in close proximity, bridged by an imidazolate ring derived from a histidyl residue. This enzyme is found in the cytosol of almost all eukaryotic cells and in some bacteria and has been proposed to function in the cell as a protective agent against the toxic effects of superoxide. It is the aim of this research to carry out a multi-faceted study of the properties of this enzyme. We will continue our physical and spectroscopic characterization of the enzyme in both its oxidized and reduced states using principally vis-uv-near-IR, ESR, and NMR spectroscopy. We will also continue to study the metal ion binding reactions of the apoprotein and characterize the metal ion-depleted and metal ion-substituted derivatives with respect to the kinetics and thermodynamics of metal-protein interactions. We will carry out studies of the enzyme designed to answer questions related to the relationship of the structure of the enzyme and its enzymatic activity as a superoxide dismutase using chemically modified derivatives of the enzyme as well as metal-substituted derivatives. Finally, we will extend our studies of the functional role of superoxide dismutases by preparation of mutant strains of yeast that contain alterations in their superoxide dismutase genes using the modern genetic techniques of in vitro mutagenesis. We believe that the results of the research proposed here will lead to a better understanding of metal ion-protein interactions in general and to the role of superoxide dismutases in the mechanism of oxygen toxicity.